Expression Protocol in M9 Minimal Media via T7 Promoter
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Expression Protocol in M9 Minimal Media via T7 Promoter
The following protocol has been used successfully to 15 N or 13 C/15 N label our proteins using our pET1120/BL21(DE3) expression system: Preparing M9 minimal media begins with preparing a 5x stock solution of M9 salts. Generally, M9 salts contain a nitrogen source in the form of NH4 Cl. Since we want to add a labeled nitrogen source, our 5x salts are prepared minus NH4 Cl. Standard 5 X M9 Minimal Media salts minus nitrogen source For 1L 5xM9 salts:- 64g Na2 HPO4 -7H2 O
- 15g KH2 PO4
- 2.5g NaCl
- H2 O to final 1L volume and autoclave
- 100mL of 5xM9 salts
- 1 mL 1M MgSO4
- 50 uL 1M CaCl2
- 5 mL 100x Basal Medium Eagle Vitamin Solution (Gibco)
- 2.5 mL filter sterilized NH4 Cl (0.2 g/mL) or 0.5g dry
- 10 mL 20% d-glucose or 2g dry
- glass distilled & autoclaved H2 O to final volume of 500mL
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pH solution to 7.3 and filter sterilize (0.2u filter).
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Introduce media to a pre-autoclaved, wide-bottom (baffled) 2L flask and add ampicillin to a final concentration of 70-100 ug/ml.
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Grow 5mL overnight culture in same media to inoculate 500mL M9.
- Shake culture at 37o C until an OD600 of 07+/-0.2 then induce protein expression with the addition of IPTG (0.01-0.1 mM final concentration).
